The purpose of this study was to learn more about the role of the retinoblastoma tumor suppressor protein (pRb) in cell proliferation, differentiation and senescence. To do so, a mammalian expression vector containing the cDNA of a temperature-sensitive (ts) mutant of pRb was prepared and transfected together with the puromycin resistance gene into mouse 3T3 pRb (-/-) fibroblasts. After several weeks of selection in puromycin-containing cell culture medium, eighteen independent 3T3 clones were isolated and expanded into the cell lines 3T3 ts pRb 1-18. These cell lines were then analyzed for expression of the ts pRb protein using immunofluorescence and Western Blot techniques. Cells were grown on coverslips and subsequently fixed, permeabilized and subjected to immunofluorescence staining for pRb with a monoclonal anti-pRb antibody and a rhodamine-conjugated secondary antibody. Three of the 3T3 cell lines were determined to be positive for ts pRb expression in this assay. In order to confirm this result, whole-cell protein extracts of all cell lines were prepared. Proteins in these extracts were separated by SDS-PAGE and the presence of pRb was determined by Western Blotting.